Metallic nanoparticles, preparation and uses thereof

ABSTRACT

The present application relates to activatable nanoparticles which can be used in the health sector, in particular in human health, to disturb, alter or destroy target cells, tissues or organs. It more particularly relates to nanoparticles which can generate a significantly efficient therapeutic effect, when exposed to ionizing radiations. The inventive nanoparticle is a metallic nanoparticle having, as the largest size, a size comprised between about 80 and 105 nm, the metal having preferably an atomic number (Z) of at least 25. The invention also relates to pharmaceutical compositions comprising a population of nanoparticles as defined previously, as well as to their uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/383,049, filed Jan. 31, 2012, now U.S. Pat. No. 9,700,621, which isthe U.S. national stage application of International Patent ApplicationNo. PCT/EP2010/059871, filed Jul. 9, 2010, which claims the benefit ofU.S. Provisional Patent Application No. 61/224,576, filed Jul. 10, 2009,the disclosures of which are hereby incorporated by reference in theirentireties, including all figures, tables and amino acid or nucleic acidsequences.

FIELD OF THE INVENTION

The present application relates to activatable nanoparticles which canbe used in the health sector, in particular in human health, to disturb,alter or destroy target cells, tissues or organs. It more particularlyrelates to nanoparticles which can generate a surprisingly efficienttherapeutic effect, when exposed to ionizing radiations such as X-Rays,γ-Rays, radioactive isotopes and/or electron beams. The inventivenanoparticle is a metallic nanoparticle having, as the largest size, asize comprised between about 80 and about 105 nm, the metal havingpreferably an atomic number (Z) of at least 25. The invention alsorelates to pharmaceutical compositions comprising a population ofnanoparticles as defined previously, as well as to their uses.

BACKGROUND

Radiations of various forms such as X-Rays, gamma-Rays, UV-Rays, laserlight, microwaves, electron beams as well as particle beams of, forexample neutrons, and protons, have been used to treat cancer relatedissues. Some of said radiations have been used in such applications, incombination with radiation sensitive molecules. Electromagnetic andionizing radiations are indeed capable in particular of breaking the DNAmolecule of the cell, thereby killing cells and/or preventing said cellfrom growing and dividing. This effect is mainly due to indirect damagescreated in particular by electrons and/or high energy photons emittedafter ionization that will be responsible for free radicals generation.

The term “Ionizing radiations” refers to highly-energetic particles orwaves that can ionize an atom or molecule. Ionizing ability depends onthe energy of individual particles or waves, and not on their number. Alarge flood of particles or waves will not, in the most-commonsituations, cause ionization if the individual particles or waves areinsufficiently energetic. A typical ionizing radiation is a radiation,the energy of which is higher than 2 KeV.

Radiosensitization by gold nanoparticles (GNPs) has been identified as apromising approach for improving radiotherapy.

U.S. Pat. No. 6,955,639 (Hainfeld et al.) describes a method ofenhancing X-Rays radiation effects using metal, in particular gold,nanoparticles, the size (diameter) of the metal core being preferably,for biodistribution reasons, in the range of 0.8 to 20 nm, morepreferably 0.8 to 3 nm.

Herold et al. (Int. J. Rad. Biol. 76 (2000) 1357) indicate that goldnanoparticles of small size (˜2 nm) should diffuse more homogeneouslythroughout the tumor mass.

Chithrani et al. (Nano Lett. 6 (2006) 662; Nano Lett. 7 (2007) 1542)showed a preferential penetration and accumulation of 50-nm diameterGNPs in Hela cells.

Chang et al. (Cancer Sci. 99 (2008) 1479) showed that, in a melanomatumour-bearing mice model, 13-nm diameter GNPs in conjunction with asingle dose of 25 Gy from a 6 MeV electron beam led to a more pronouncedreduction of the tumor volume than in the control groups.

Zhang et al. (Biomed Microdevices (2009), 11:925-933) provides in silicodatas (Monte Carlo simulation model) confirming that gold nanoparticlescan enhance the effective dose of radiation, but does not study thenanoparticle's size impact on such dose enhancement. This documentrefers to the 1.9 nm diameter-nanoparticles of Hainfeld in the contextof radiation therapy (see page 930, right column), but provides noresult which may be of help to accurately quantify a dose enhancementfactor in a biological system (see Montenegro et al., J. Phys. Chem. A.2009, 113, 12364-12369: “Monte Carlo Simulations and Atomic Calculationsfor Auger Processes in Biomedical Nanotheranostics”), in particular in ahuman being.

Inventors herein provide powerful nanoparticles, which are surprisinglyable to achieve a more efficient perturbation, alteration or destructionof target cells in vitro, ex vivo and in vivo when said nanoparticlesare exposed to ionizing radiations, than nanoparticles described in theprior art, as herein demonstrated.

The inventive nanoparticle is a metallic nanoparticle havingadvantageously, as the largest size, a size comprised between about 80nm and about 105 nm, the nanoparticle being made of a metal havingpreferably an atomic number (Z) of at least 25. The advantageousproperties of the herein described nanoparticles could not beextrapolated from the art which, in contradiction with the presentinvention, suggests the use of gold nanoparticles of small size toincrease the dose enhancement factor [see in particular Brun et al.(Colloids and Surfaces B: interfaces, 72 (2009) 128-134: “Parametersgoverning gold nanoparticle X-ray radiosensitization”) who reveal theinfluence of the gold nanoparticles concentration]. The resultsappearing on FIG. 4(B) of Brun et al. in particular, reveal a doseenhancement factor increase when the size of gold nanoparticledecreases, for a given gold concentration (the gold content varying withthe gold nanoparticle radius according to a factor 3).

For a given metal concentration and a given ionizing radiationabsorption ability, the metallic nanoparticles herein described, thesize of a typical metallic nanoparticle being preferably between about80 nm and about 105 nm, are responsible for an increased therapeuticefficacy (ability to generate target cells damages) when compared tonanoparticles of smaller sizes, in particular when compared tonanoparticles having a size of 60 nm or less.

For a given metal concentration and an equivalent X-Rays attenuation atcellular level, the metallic nanoparticles herein described exhibit astronger ability to kill cells and/or prevent their division.

Another feature exhibited by the herein described nanoparticles, istheir ability, when exposed to ionizing radiations, to generate atherapeutic effect when in contact with target cells. In other words,the therapeutic efficiency observed under irradiation does not requirethe nanoparticles cell uptake. Such a property is herein described forthe first time.

Indeed, until now, the target cell uptake was believed, in the art, tobe required for the nanoparticles to be able to generate efficientcellular lethal damages under irradiation (see for example Kong et al.(Small 4 (2008) 1537)).

The present invention thus goes against the prejudice of the all priorart leading the skilled person, mainly for biocompatibility,biodistribution, and cell uptake reasons, to the use, in terms ofmedical applications, of nanoparticles with a diameter from about 1 to20 nm, at most 60 nm, with a particular and long-lasting interest for50-nm nanoparticles (see for example Chithrani et al. (2006) and Changet al. (2008)).

The nanoparticles of the present invention further advantageously allowa reduction in the amount of metal to be administered to a subject, aswell as a reduction in the number of nanoparticles administration steps,to a minimum, in the context of a complete radiotherapeutic treatmentprotocol, thereby favouring their tolerance by the subject.

SUMMARY OF THE INVENTION

Inventors have now discovered that it is possible to disturb, alter ordestroy target cells, tissues or organs, in particular abnormal cells ortissues, herein defined as benign cells or tissues, or diseased cells ortissues, such as pre-malignant or malignant cells (cancerous cells) ortissues (tumors), superficial or deep in the body, with a surprisinglyincreased efficiency, using a nanoparticle made of a metal havingpreferably an atomic number (Z) of at least 25, the largest size of saidnanoparticle being comprised between about 80 nm and about 105 nm.

It is an advantage of the present invention to provide nanoparticlesthat are not noxious by themselves but can be safely employed, inappropriate conditions, to functionally disturb, alter or destructtarget cells in an animal, preferably in a mammal, even more preferablyin a human. The desired therapeutic effect of nanoparticles is indeedstrictly dependent from their ionization, said ionization beinggenerated by an ionizing radiation source which is itself advantageouslycontrolled, in terms of quality and quantity, and used in a targeted,i.e., localized, way, by the man of the art.

The present invention indeed describes nanoparticles which can induce acell perturbation, alteration or destruction in vitro, ex vivo or invivo when said cell is exposed to ionizing radiations such as inparticular X-Rays, gamma-rays (γ-Rays), radioactive isotopes, ion beamsand/or electron beams.

The herein described nanoparticles are able to directly interact withincoming radiations and to generate a surprisingly efficient therapeuticeffect, without it being necessary for the nanoparticles to beinternalized by the target cells.

The nanoparticles according to the present invention can be covered witha biocompatible coating preferably favouring the nanoparticle stabilityin a physiological fluid as further described herein below.

The present nanoparticles can be used, if appropriate and preferred, ina targeted manner using for example a surface component enablingspecific targeting of biological tissues or cells. They however do notrequire a targeting molecule to concentrate into the target cells ortissues.

The Enhanced Permeation and Retention (“EPR”) effect is indeedresponsible for passive accumulation into the tumor mass, after a giventime following injection by the intravenous route (one possible route ofadministration) of the nanoparticles. It has indeed been observed thatthe tumor vessels are quite distinct from normal capillaries and thattheir vascular “leakiness” encourages selective extravasation ofnanoparticles not usual in normal tissues. The lack of effective tumourlymphatic drainage prevents clearance of the penetrant nanoparticles andpromotes their accumulation. The present nanoparticles are thus able tosuccessfully target primary as well as metastatic tumors afterintravenous administration.

The present nanoparticles can also be advantageously administeredthrough intratumoral, or intra-arterial route.

It is therefore an object of the present invention to use a nanoparticleaccording to the present invention or a population of such nanoparticlesto alter or destroy a target cell, tissue or organ.

Particular embodiments herein disclosed relate to the use of apopulation of metallic nanoparticles to prepare a pharmaceuticalcomposition intended to perturb, alter or destroy target mammalian cellswhen said cells are exposed to ionizing radiations, wherein thenanoparticles are made of a metal having an atomic number (Z) of atleast 25 and the mean largest size of the nanoparticles of thepopulation is between about 80 and 105 nm, and to the correspondingmethods of treatment. Products according to the present invention, inparticular nanoparticle and population of metallic nanoparticles, foruse in the treatment of cancer, are in particular herein disclosed.

Another embodiment is based on a pharmaceutical composition, inparticular, as will be apparent from the all description, apharmaceutical composition intended to perturb, alter or destroy targetcells in a mammal when said cells are exposed to ionizing radiations,said pharmaceutical composition comprising a population of metallicnanoparticles, as herein defined, and a pharmaceutically acceptablecarrier or excipient, wherein the nanoparticles are made of a metalhaving an atomic number (Z) of at least 25 and the mean largest size ofthe nanoparticles of the population is between about 80 and 105 nm.

Another embodiment relates to the use of a nanoparticle, a population ofmetallic nanoparticles or composition according to the presentinvention, to prevent or treat a cancer or to alleviate the symptoms ofa cancer in an animal, when said animal is exposed to radiations, inparticular to ionizing radiations as herein defined.

The present disclosure in particular encompasses a method for preventingor treating a cancer or for alleviating the symptoms of a cancer in asubject, the subject being an animal, in particular a mammal, preferablya human, by administering a metallic nanoparticle, a population ofmetallic nanoparticles or a composition comprising a population of suchnanoparticles according to the present invention, to the subject, andexposing said subject to radiations, in particular to ionizingradiations.

In another aspect, the present disclosure provides kits comprising anyone or more of the herein-described products, i.e., nanoparticles andcompositions, together with a labeling notice providing instructions forusing the product(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B:

FIG. 1A shows the Transmission Electronic Microscopy (TEM) images of thegold nanoparticles described in Table 1 (see example 1).

FIG. 1B shows the size distribution of the gold nanoparticles (GNPs)described in Table 1.

FIGS. 2A and 2B: The crystalline structure of the as prepared goldnanoparticle is determined by electronic diffraction.

FIG. 2A shows the electronic diffraction pattern of referencenanoparticles (gold nanoparticles with Cubic Face Center structure areused as reference to establish the camera constant (Lλ) of thetransmission electronic microscope) and of gold nanoparticles (GNPs)from example 1.

FIG. 2B reports the indexation of the gold nanoparticles (from example1), electronic diffraction pattern showing a Cubic Face Center (CFC)structure of the gold nanoparticles.

Indexing the electronic diffraction pattern consists in the followingsteps:

-   1) Establishing the camera constant from electronic diffraction    pattern of the reference,-   2) Measuring the ring diameter (D1, D2, Dn) of electronic    diffraction pattern of the gold nanoparticles from example 1,-   3) Calculating the d_(hkl), using the expression d_(hkl)=L*λ/(Dn/2),-   4) Using existing structure data base to index each ring.

FIGS. 3A and 3B:

FIG. 3A shows clonogenic survival assays using HT29 colon cancer cellsirradiated with a 200 KVp X-Ray energy beam, in the absence (negativecontrol) or in the presence of 12 μM, 20 μM and 130 μM of gold atcellular level for gold nanoparticles with a particle size of 15 nm(GOLD-15 from example 1). Irradiation dose varies from 0 (noirradiation) to 4 Gy.

Negative control with HT29: square dots

Gold-15 nanoparticles with HT29 12 μM: cross dots

Gold-15 nanoparticles with HT29 20 μM: triangle dots

Gold-15 nanoparticles with HT29 130 μM: circle dots

FIG. 3B shows clonogenic survival assays using HT29 colon cancer cellsirradiated with a 200 KVp X-Ray energy beam, in the absence (negativecontrol) or in the presence of 17 μM, 52 μM and 119 μM of gold atcellular level for gold nanoparticles with a particle size of 80 nm(GOLD-80 from example 1). Irradiation dose varies from 0 (noirradiation) to 4 Gy.

Negative control with HT29: square dots

Gold-80 nanoparticles with HT29 17 μM: cross dots

Gold-80 nanoparticles with HT29 52 μM: triangle dots

Gold-80 nanoparticles with HT29 119 μM: circle dots

FIGS. 4A and 4B: Effect of gold nanoparticles size on the DoseEnhancement Factor (DEF) for similar gold concentration at cellularlevel, also herein identified as gold concentration per target cell.

FIG. 4A

-   -   Gold (Au) concentration at cellular level is expressed in μM.    -   Gold concentration below 20 μM ([Au]<20 μM): cross dots    -   Gold concentration between 20 μM and 83 μM (20 μM≤[Au]≤83 μM):        diamond dots    -   Gold concentration between 95 μM and 148 μM (95 μM≤[Au]≤148 μM):        square dots    -   Gold concentration at 400 μM ([Au]=400 μM): triangle dot FIG. 4B    -   Gold (Au) concentration per target cell is expressed as follows:        Cell:Au=1:X (X is expressed in nmole)        Cell:Au≤1:15×10⁻⁵:cross dots        1:20*10⁻⁵≤Cell:Au≤1:45×10⁻⁵:diamond dots        1:60*10⁻⁵≤Cell:Au≤1:110×10⁻⁵:square dots        Cell:Au=1:182×10⁻⁵:triangle dot

FIGS. 5A and 5B:

FIG. 5A shows a threshold effect for a gold nanoparticle size ≥80 nm.The gold concentration at cellular level was between 20 μM and 83 μM.

The corresponding gold concentration per target cell was between 20×10⁻⁵nmole and 45×10⁻⁵ nmole.

Two linear tendency curves are established with a significant differencebetween their respective slope values: gold nanoparticles with a sizebetween 15 nm and 60 nm present a linear tendency curve with a slope of0.0033. Gold nanoparticles with a size between 80 nm and 105 nm presenta linear tendency curve with a slope of 0.0384. The slopes ratiorevealed by said two curves is of about 10. The threshold effectobserved when considering DEF is induced by the metallic nanoparticlesize when said size is of about 80 nm or more.

FIG. 5B shows a threshold effect for gold nanoparticle size ≥80 nm. Thegold concentration at cellular level was between 95 μM and 148 μM.

The corresponding gold concentration per target cell was between 60×10⁻⁵nmole and 110×10⁻⁵ nmole.

Two linear tendency curves are established with a significant differencebetween their respective slope values: Gold nanoparticles with a sizebetween 15 nm and 60 nm present a linear tendency curve with a slope of0.0025. Gold nanoparticles with a size between 80 nm and 105 nm presenta linear tendency curve with a slope of 0.0865. The slopes ratiorevealed by said two curves is of about 30. The threshold effectobserved when considering DEF is induced by the metallic nanoparticlesize when said size is of about 80 nm or more.

FIGS. 6A and 6B:

FIG. 6A shows the X-ray attenuation as a function of gold concentrationfor each gold nanoparticle described in example 1, Table 1.

HU value as a function of [Au] (g/L) for GOLD-15: diamond dots

HU value as a function of [Au] (g/L) for GOLD-30: square dots

HU value as a function of [Au] (g/L) for GOLD-60: triangle dots

HU value as a function of [Au] (g/L) for GOLD-80: cross dots

HU value as a function of [Au] (g/L) for GOLD-105: + dots

FIG. 6B shows the impact of gold nanoparticle size on X-Raysattenuation. The slope for each size of gold nanoparticle is obtainedfrom FIG. 6A and reported as a function of gold nanoparticle size.

FIGS. 7A and 7B:

FIG. 7A shows the surviving fraction under a 4 Gy irradiation (SF4) ofHT29 cells incubated with gold nanoparticles (GOLD-60 from example 1)for less than 5 minutes.

FIG. 7B shows the surviving fraction under a 4 Gy irradiation (SF4) ofHT29 cells incubated with gold nanoparticles (GOLD-60 from example 1)for about 12 hours.

DETAILED DESCRIPTION OF THE INVENTION

It has been surprisingly found by inventors that a nanoparticle made ofa metal, preferably a metal having an atomic number (Z) of at least 25,and having, as the largest size, a size comprised between about 80 and105 nm, can substantially enhance the therapeutic effect of a localirradiation intended to disturb, alter or destroy abnormal cells,tissues or organs in an animal.

A strong enhancement of radiotherapy efficacy can be observed for thefirst time using metallic nanoparticles according to the presentinvention (cf. FIGS. 4A, 4B, 5A and 5B for example).

In the spirit of the invention, the term “nanoparticle” refers toproducts, in particular synthetic products, with a size in the nanometerrange.

In the context of the present invention, the term size refers to thelargest dimension of the metallic core of the nanoparticle. Typically,the largest dimension is the diameter of a nanoparticle of round orspherical shape, or the longest length of a nanoparticle of ovoid oroval shape.

The nanoparticle's shape can be for example round, flat, elongated,spherical, ovoid or oval, and the like. The shape can be determined orcontrolled by the method of production, and adapted by the person of theart according to the desired applications.

As the shape of the particles can influence their “biocompatibility”,particles having a quite homogeneous shape are preferred. Forpharmacokinetic reasons, nanoparticles being essentially spherical,round or ovoid in shape are thus preferred. Spherical or round shape isparticularly preferred.

The largest size of the nanoparticles according to the invention isadvantageously, as demonstrated by the experimental part, comprisedbetween about 70 nm and about 130 nm, advantageously between about 75 or80 nm and about 105 nm, preferably between about 75 nm and about 95 nmor between about 80 nm and about 90 or 95 nm.

Inventors surprisingly demonstrate for the first time that thenanoparticle penetration into the target cell, a tumor cell for example,is not required, in the context of the present invention, to perturb,destroy or alter said cell. Indeed, an equivalent effect on target cellshas been observed by inventors in both conditions wherein nanoparticleshave been incorporated by target cells or are in contact, in particularexternal contact, with said cells.

Inventors herein describe the use of a metallic nanoparticle as hereindisclosed, or of a population of such metallic nanoparticles, to preparea pharmaceutical composition intended to perturb, disturb, alter ordestroy target mammalian cells when said cells are exposed to ionizingradiations. In said population, the nanoparticles are made of a metal,the metal having preferably an atomic number (Z) of at least 25.Advantageously, the mean largest size of the nanoparticles of thepopulation is between about 70 nm and about 130 nm, advantageouslybetween about 75 or 80 nm and about 105 nm, preferably between about 75nm and about 95 nm or between about 80 nm and about 90 or 95 nm.

In a typical population of nanoparticles, as referred to previously,constituted by nanoparticles obtained according to a method of the art(as further described below), wherein the mean largest size of ananoparticle of the population is between about 80 nm and about 105 nm,the largest size of a nanoparticle of the population is comprisedbetween about 60 nm and 155 nm, typically between 60, 65, 70, 75, or 80,and, 105, 110, 130, 140, 150 or 155 nm.

In other words, 95% (2σ) of the population is made of nanoparticles thelargest size of which is between about 60 nm and about 155 nm or 68%(1σ) of the population is made of nanoparticles the largest size ofwhich is between about 70 nm and about 130 nm.

The metallic nanoparticles according to the present invention are madeof a metal, said metal having preferably an atomic number of at least25, advantageously at least 40 or 50, more preferably at least 60 or 70.

Such a metal may be selected from gold (Au—Z=79), silver (Ag—Z=47),platinum (Pt—Z=78), palladium (Pd—Z=46), tin (Sn—Z=50), tantalum(Ta—Z=73), ytterbium (Yb—Z=70), zirconium (Zr—Z=40), hafnium (Hf—Z=72),terbium (Tb—Z=65), thulium (Tm—Z=69), cerium (Ce—Z=58), dysprosium(Dy—Z=66), erbium (Er—Z=68), europium (Eu—Z=63), holmium (Ho—Z=67), iron(Fe—Z=26), lanthanum (La—Z=57), neonydium (Nd—Z=60), praseodynium(Pr—Z=59), and mixtures thereof.

The metal is preferably selected from gold (Au), silver (Ag), platinum(Pt), palladium (Pd), tin (Sn), zirconium (Zr) and iron (Fe).

The atomic number (also known as the proton number) is the number ofprotons found in the nucleus of an atom. It is traditionally representedby the symbol Z. The atomic number uniquely identifies a chemicalelement. In an atom of neutral charge, the atomic number is equal to thenumber of electrons.

Z participates to the incoming radiations absorption capacity ofnanoparticles.

In a preferred embodiment of the present invention, the metallicnanoparticles are made of gold.

In the present invention, mixture of metals as identified previously ina particular nanoparticle, or in a particular population ofnanoparticles, is also possible.

Nanoparticles having a low specific surface area (SSA) are furtherpreferred in order to limit their interactions with the surroundingenvironment.

The Specific Surface Area (SSA) is a material property of solids whichmeasures the total surface area per unit of mass (m²/g). SSA appears tobe an important factor affecting the nanoparticle biological systeminterface; on an equal mass-dose basis, it has been reported thatultrafine particles cause more adverse effects, such as inflammation,when administered in an animal, than do fine particles (see for exampleNel et al. (Nature Materials 8 (2009) 543).

Metallic nanoparticles, the largest size of which is between about 80and 105 nm are particularly advantageous regarding SSA as explainedbelow.

For the purpose of the present invention, the nanoparticle specificsurface area is for example comprised between about 1 m²/g and 50 m²/g.The specific surface area is preferentially comprised between 2 m²/g and20 m²/g.

The specific surface area of a spherical nanoparticle may be estimatedusing the following equation (SSA=3000/(d×r)), d being the density ofthe metallic nanoparticle, and r the radius of the nanoparticle.

Hence spherical gold nanoparticles with a particle size of 15, 30, 60,80 and 100 nm will develop a specific surface area respectively of 20.7,10.3, 5.2, 3.9 and 3.1 m²/g, for a gold nanoparticle density of 19.32.

Spherical iron nanoparticles with a particle size of 15, 30, 60, 80 and100 nm will develop a specific surface area respectively of 50.8, 25.4,12.7, 9.5 and 7.6 m²/g, for an iron nanoparticle density of 7.87.

Inventors herein disclose that the surprising efficiency of thenanoparticles according to the present invention is mainly due to theirsize. A nanoparticle, the size of which is of at least 80 nm, is indeed,when exposed to ionizing radiations, capable of generating more damagesto target cells than is a nanoparticle of smaller size, in particular ananoparticle of 60 nm or less. Inventors thus herein highlight thefundamental and direct influence of the nanoparticle size on celldisturbance under ionizing radiations, the herein described sizesfavouring therapeutic applications in a mammal, when said size reaches,and preferably exceeds, the 80 nm threshold (see experimental part). Thelargest the nanoparticle size in the herein identified range of sizes,the more efficient is their ability to generate cell damages.

A possible explanation of this mechanism could be due to thenanoparticle ability to deliver the captured energy (ionizing radiation)in a better or different way.

In example 2 and 3, in order to differentiate the influence of goldnanoparticle size from the influence of gold concentration on thetherapeutic effect induced by the nanoparticles under irradiation, invitro assays were all performed by inventors using the gold nanoparticlesize as unique adjustable parameter. The experimental results obtainedby inventors revealed the surprising influence of the nanoparticle size(for a constant metal concentration) on the amplification of thetherapeutic efficacy (ability to kill cells and/or to prevent cells todivide).

In order to produce an efficient therapeutic effect under ionizingradiations, the use of metallic nanoparticles with a nanoparticle size80 nm requires a metal concentration per target cell which is betweenabout 2 and 7 times, in particular between 4 and 7 times or between 2and 5 times, inferior to the metal concentration per target cellrequired when using metallic nanoparticles with a nanoparticle size ofabout 60 nm or less (see examples 2 and 3 regarding GNPs).

A significant and advantageous reduction of the metal amount to beadministered to the patient with a similar treatment efficiency,associated to a reduction of deleterious side effects, are therefore nowpossible thanks to the present invention.

The present invention further allows a significant reduction of thenumber of administration steps in the context of a radiotherapeutictreatment in particular, typically in the course of a multi fractionatedprotocol of irradiation as performed in clinic until now. Indeed, thenanoparticles described in the present patent application are largeenough to favour their retention in a tumor tissue. A substantiallydecreased target tissue clearance of the metallic larger nanoparticlessize has been observed in the literature (CHANG et al., Cancer Sci. 99(2008) 1479; Hainfeld et al., Phys. Med. Biol 49 (2004) N309).

The required doses of ionizing radiations are preferably doses comprisedbetween about 0.05 Gray and about 16 Grays, preferably between about0.05 Gray and about 6 Grays, for applications performed in vitro.

Doses are comprised between more than about 0.05 Gray and less thanabout 16 or 30 Grays for applications performed, in particular locally,ex vivo or in vivo.

Total ionizing radiations range from about 1.5 Gray up to about 85 Graysin the human according to the current practice. Additional irradiationboost of about 40 Gray may also be provided in the human, according tothe current practice.

The total dose of radiations delivered can be given following differentschedules such as single dose, fractionated doses, hyperfractionateddoses, etc.

Irradiated nanoparticles herein described provide, as demonstrated inthe experimental section, a clear therapeutic effect improvement whencompared to the effect obtained using irradiated nanoparticles ofsmaller sizes.

The nanoparticles according to the present invention are advantageouslybiocompatible, that is to say, they can be safely administered to ananimal organism, typically a mammal, in particular a human, to providetheir therapeutic effect. Said biocompatibility can be ensured forexample by the nature of the metal(s) constituting the particle and/orby an optional coating.

Preferred nanoparticles according to the invention are covered with abiocompatible coating regardless of the route of administration. Whenthe nanoparticles of the present invention are administered to a subjectvia the intravenous (IV) route, such a biocompatible coating isparticularly advantageous to optimize the biodistribution ofnanoparticles in the context of the previously described EPR effect. Afull biocompatible coating of the nanoparticle is preferred, inparticular in the IV context, in order to avoid interaction of theparticle surface with any recognition element (macrophage, opsonins,etc.). The “full coating” implies the presence of a very high density ofbiocompatible molecules able to create at least a complete monolayer onthe surface of the particle. Said coating is responsible for the socalled “stealth effect” of the nanoparticle.

The biocompatible coating allows or favours (depending on the selectedmetal the nanoparticle is made of) in particular the nanoparticlestability in a biocompatible suspension, such as a physiological fluid(blood, plasma, serum, etc.), any isotonic media or physiologic medium,for example media comprising glucose (5%) and/or NaCl (0.9%), which isrequired for a pharmaceutical administration.

Such a biocompatible coating is obtained by treating the nanoparticlewith a surface treating agent.

Stability may be confirmed by dynamic light scattering of the metallicnanoparticles in biocompatible suspension or by ICP-MS quantification ofmetal element prior and/or after filtration, of the metallicnanoparticles in biocompatible suspension, on a 0.22 μm filter.

Advantageously, said coating preserves the integrity of the particles invivo, ensures or improves the biocompatibility thereof, and facilitatesan optional functionalization thereof (for example with spacermolecules, biocompatible polymers, targeting agents, proteins, etc.). Aparticular nanoparticle according to the present invention indeedfurther comprises a surface component enabling specific targeting ofbiological tissues or cells. Such a surface component is preferably atargeting agent allowing the nanoparticle interaction with a recognitionelement present on the target cell. Such targeting agents can act oncethe nanoparticles are accumulated in the tumor. As the conformation ofthe targeting agent will be responsible for its interaction with thetarget, the density of said targeting agent is to be controlledcarefully. A high density thereof can indeed perturb the targeting agentconformation and in consequence its recognition by the target cell (seefor example J A Reddy et al. Gene therapy 9 (2002) 1542; Ketan B.Ghaghada et al. Journal of Controlled Release 104 (2005) 113). Inaddition, a high target agent density may favour nanoparticles clearanceby the Reticulo Endothelial System (RES) during circulation in thevasculature.

The biocompatible coating can be composed of any amorphous orcrystalline structure.

In general, the coating can be non-biodegradable or biodegradable. Bothoptions can be used for the purpose of the present invention.

Examples of non-biodegradable coatings are one or more materials orsurface treating agents selected in the group consisting of silica,alumina, sugar (agarose for example), phosphate, silane, thiol,zwitterionic compounds, lipids, saturated carbon polymers (polyethyleneoxide for example) and inorganic polymers, reticulated or not, modifiedor not (polymethacrylate or polystyrene for example), as well ascombinations thereof.

Examples of biodegradable coatings are for example one or more materialsor surface treating agents selected from the group consisting of abiological molecule, modified or not, natural or not and a biologicalmolecular polymer; modified or not, of natural shape or not. Thebiological polymer may be a phospholipid, a saccharide, anoligosaccharide or a polysaccharide, polysulfated or not, for exampledextran.

The aforementioned materials, compounds or surface treating agents canbe used alone or in combinations, mixtures or assemblies, composite ornot, covalent or not, optionally in combination with other compounds.Moreover, it is also possible to use any one of the aforementionedmaterial, said material being naturally water-soluble or lipid-solubleor being artificially modified to become water-soluble or lipid-soluble.

The biocompatible coating preferably comprises or is made of a compoundselected in the group consisting of an inorganic agent, an organicagent, and a mixture or combination thereof.

Appropriate inorganic agent may be selected from the group consisting ofan oxide, a hydroxide, and an oxyhydroxide. The inorganic agent maycomprise for example silicium, aluminium, calcium and/or magnesium.

Such agents can be used to charge the nanoparticle either positively ornegatively in order to modulate interactions of said nanoparticle withthe biological media.

An inorganic agent selected from the group consisting of, for examplemagnesium and calcium, will bring a positive charge to the surface ofthe nanoparticle at a pH of 7.

For example, the silicium may be used to bring a negative charge to thesurface of the nanoparticle at a pH of 7.

An appropriate organic agent may be any agent comprising a functioncapable of interacting with a nanoparticle according to the presentinvention and a function conferring biocompatibility to saidnanoparticle.

The agent comprising a function capable of interacting with ananoparticle may be for example a carboxylate (R—COO⁻), a silane(R—Si(OR)₃), a phosphonic function (R—PO(OH)₂), a phosphoric function(R—O—PO(OH)₂), or a thiol function (R—SH).

The agent comprising a function capable of conferring biocompatibilityto a nanoparticle according to the present invention may have a stericfunction and/or an electrostatic function. Such agent with a stericfunction may be selected from the group consisting of polyethyleneglycol (PEG), polyethylenoxide, Polyvinylalcohol, Polyacrylate,Polyacrylamide (poly(N-isopropylacrylamide)), Polycarbamide, abiopolymer or polysaccharide such as Dextran, Xylan, cellulose,collagene, and a zwitterionic compound such as polysulfobetain, etc.

Agent with a positive electrostatic function may be an amine such asaminopropyltriethoxisilane, polylysine or 2-aminoethanethiol.

Agent with a negative electrostatic function may be selected from thegroup consisting of phosphate (for example a polyphosphate, ametaphosphate, a pyrophosphate, etc.), carboxylate (for example citrateor dicarboxylic acid, in particular succinic acid) and thiol (forexample a carboxy terminated thiol such as mercaptosuccinic acid).

The coating can also contain different functional groups (or linkersegments), allowing any molecule of interest to bind to the surface ofthe particle, such as a surface component enabling specific targeting ofbiological tissues or cells.

A typical example of a nanoparticle according to the invention is ananoparticle made of gold. Such a gold nanoparticle can comprise, inaddition, as a biocompatible coating, a coating made of thiol compoundssuch as polyethyleneglycol-thiol (PEG-SH), thioglucose, or carboxylatecompounds such has citrate.

Another example of a nanoparticle according to the invention is ananoparticle made of gold comprising, as a biocompatible coating, acoating made of thiol agents bearing at least one functional groupselected from polyethylene, amine or carboxyl, or a coating consistingin citrate.

The present nanoparticles offer the advantage of being easy to prepare.Methods of producing metallic nanoparticles are indeed well known in theart (see for example Brian L. Cushing et al. (Chem. Rev. 104 (2004)3893). Typically, metallic nanoparticles are obtained by precipitationof a metallic element in an aqueous or a non-aqueous solution, saidprecipitation involving chemical reduction of the metallic cation.Another possible way of production of metallic nanoparticles is throughradiation-assisted reduction.

Another object of the invention relates to a method of producing ametallic nanoparticle or population of metallic nanoparticles such asdefined hereinabove, comprising:

-   -   providing a metallic element as herein identified, preferably a        metallic element having an atomic number (Z) equal to or above        25,    -   preparing the metallic nanoparticle from said metallic element        by precipitation of said metallic element in a medium, in the        presence of a reducing agent, and, optionally    -   adding a complexing agent to the medium (the complexing agent        being added prior to, during or after the addition of the        reducing agent), the reducing agent and the complexing agent        being optionally the same compound, and, optionally,    -   coating the nanoparticle using a surface treating agent as        described previously.

A medium typically used in the present invention may be selected from anaqueous solution, an alcoholic solution, etc.

A reducing agent typically used may be selected from citrate, ascorbicacid, 2-mercaptosuccinic acid.

A complexing agent typically used may be selected from citrate, thiolsuch as 2-mercaptosuccinic acid, etc.

The coating step advantageously consists in placing the nanoparticle incontact with a surface treating agent as defined previously.

In a particular embodiment, a method of producing a population ofnanoparticles comprises the following steps, preferably in order:

-   a) providing, as a precursor, a metallic element as herein    identified, preferably a metallic element having an atomic    number (Z) equal to or above 25,-   b) Precipitating the precursor of step a) in a polar medium as    defined previously in the presence of a reducing compound, by    preferably adjusting the precursor and/or the reducing compound    concentration and/or the temperature,-   c) optionally adding a complexing agent in the polar medium, prior    to, during or after the precipitation step b), the complexing agent    and the reducing agent being optionally the same compound,-   d) optionally washing the suspension obtained at the end of step b)    or c) to remove any impurities, reducing agent and/or complexing    agent,-   e) optionally concentrating the suspension obtained at the end of    step d), and-   f) optionally coating the nanoparticles.

The population described above may be further submitted to a formulationstep before being administered to a subject.

In a particular example, a method of producing a nanoparticle accordingto the present invention, the nanoparticle being made of a metal,preferably comprises the following steps in order:

-   a) precipitating a solution of gold chloride precursor (such as in    particular HAuCl₄ or KAuCl₄) in aqueous solution in a presence of a    reducing agent (such as citrate), the temperature of the medium    being comprise between 50° C. and 100° C.,-   b) optionally washing the obtained metallic nanoparticle suspension    to remove any impurities,-   c) optionally concentrating the metallic nanoparticles suspension    thus obtained,-   d) optionally coating said metallic nanoparticles by placing them in    contact with a surface treating agent as defined previously.

Another object of the invention is based on any composition comprisingnanoparticles such as defined hereinabove and/or which can be obtainedby the methods herein described. While not mandatory, the particles inthe inventive compositions advantageously have quite homogeneous shapeas indicated previously.

Biocompatible suspensions comprising a high concentration of metalelement (300 g/L for example) can be obtained with a method as hereindescribed.

A particular object of the invention relates to a pharmaceuticalcomposition comprising nanoparticles such as defined hereinabove and,optionally, a pharmaceutically acceptable excipient or vehicle.

The compositions can be in the form of a solid, liquid (particles insuspension), aerosol, gel, paste, and the like. Preferred compositionsare in the form of an injectable formulation, preferably in a liquidform.

The excipient or vehicle which is employed can be any classical supportfor this type of application, such as for example saline, isotonic,sterile, buffered solutions, and the like. They can also comprisestabilizers, sweeteners, surfactants, and the like. They can beformulated for example as ampoules, aerosol, bottles, tablets, capsules,by using known techniques of pharmaceutical formulation.

Advantageously, the metal concentration to be administered per targetcell is between about 10⁻⁷ nmole (Cell:[metal]=1:10⁻⁷ (nmole)) and about5×10⁻¹ nmole (cell:[metal]=1:5×10⁻¹ (nmole)). More preferably, the metalconcentration per target cell is between about 10⁻⁶ nmole(Cell:[metal]=1:10⁻⁶ (nmole)) and about 2×10⁻¹ nmole(cell:[metal]=1:2×10⁻¹ (nmole)).

Even more preferably, the metal concentration per target cell is betweenabout 10⁻⁶ nmole (Cell:[metal]=1:10⁻⁶ (nmole)) and about 10⁻³ nmole(cell:[metal]=1:10⁻³ (nmole)) or between about 10⁻⁶ nmole(Cell:[metal]=1:10⁻⁶ (nmole)) and about 10⁻⁴ nmole (cell:[metal]=1:10⁻⁴(nmole)).

In the herein described compositions, appropriate or desirableconcentrations of metal are comprised between about 1 mg and about 100mg of metal per gram of target mammalian cells, such as, in particular,tumor mammalian cells, typically between about 1 mg or 5 mg and 50 mg ofmetal per gram of target mammalian cells.

Generally, the compositions in liquid form comprise between 0.01 g/L and300 g/L of metal, preferably at least 1 g/L, 5 g/L, 10 g/L, 20 g/L, 40g/L, 60 g/L, 80 g/L, 100 g/L, 150 g/L, 200 g/L or 250 g/L.

Metal quantification is ideally performed by ICP-MS.

The above identified metal concentrations vary depending of the patientsubject, the selected route of administration, the nature of the targetcells, etc., and are easily adjustable by the man of the art.

The nanoparticles, populations of nanoparticles and compositions of theinvention are products which can be used in many fields, particularly inhuman or veterinary medicine.

It is an object of the present invention to use a product as hereindescribed to alter, destroy a target cell, tissue or organ.

Depending on the energy of ionizing radiations, the particles can enableperturbation of cells and/or tissues, or destruction thereof.

Hence a particular object of the invention is based on the use of ametallic nanoparticle or a population of nanoparticles according to thepresent invention to prepare a pharmaceutical composition intended toalter, destroy target cells in an animal, when said cells are exposed toradiations, in particular to ionizing radiations, and on thecorresponding therapeutic methods.

The pharmaceutical composition can further comprises an additionaltherapeutic compound, distinct from a nanoparticle or of a population ofnanoparticles as herein described, also intended to treat cancer.

Another particular object of the invention is based on a method forinducing or causing the perturbation, lysis, apoptosis or destruction oftarget cells, in vitro, ex vivo or in vivo, comprising contacting cells,in particular target cells, with one or more nanoparticles such asdefined hereinabove, during a period of time sufficient to allow thenanoparticles to interact with said cells and, exposing the cells toradiations, appropriate radiations being in particular ionizingradiations, preferably X-Rays, γ-Rays, radioactive isotopes and/orelectron beams, said exposure inducing or causing the perturbation,lysis, apoptosis or destruction of said target cells.

The target cells can be any pathological cells, that is to say, cellsinvolved in a pathological mechanism, for example proliferative cells,such as tumor cells, stenosing cells (fibroblast/smooth muscle cells),or immune system cells (pathological cell clones). A preferredapplication is based on the treatment (for example the destruction orfunctional alteration) of malignant cells or tissue.

In this regard, a particular object of the invention is based on the useof a nanoparticle, or a population of such nanoparticles, as definedhereinabove, for producing a pharmaceutical composition intended for thetreatment in particular of a cancer, when used in combination withionizing radiations (as defined previously).

The present disclosure further encompasses the use of a composition,nanoparticle or population of nanoparticles such as defined hereinaboveto prevent or treat a cancer or to alleviate the symptoms of a cancer inan animal, when said cells are exposed to radiations, in particular toionizing radiations as defined previously.

Another particular object of the invention is based on a method forinducing or causing the perturbation, lysis or destruction of targetcells, in particular cancer cells, in vitro, ex vivo or in vivo,comprising contacting target cells with one or more nanoparticles suchas defined hereinabove, during a period of time sufficient to allow theparticles to interact with said cells, and, exposing the cells toradiations, in particular to ionizing radiations as defined previously,said exposure inducing or causing the perturbation, lysis or destructionof said cells.

Another object of the invention relates to a method for preventing ortreating a disorder, in particular a cancer, or alleviating the symptomsof the disorder, in a subject or patient, comprising administering tothe patient suffering from the disorder a nanoparticle, a population ofnanoparticles or a composition such as defined hereinabove, inconditions allowing the nanoparticles to interact (be in contact) withthe abnormal cells, in particular cancer cells, and subsequentlytreating the subject by exposing said subject to ionizing radiations,such irradiation leading to an alteration, disturbance or functionaldestruction of the patient's abnormal cells, thereby preventing ortreating a cancer.

Classical cancer management systematically implies the concurrence ofmultimodality treatments (combination of radiotherapy and chemotherapyfor example).

The herein described nanoparticles submitted to ionizing radiations, inthe context of radiotherapy, can be used in association with a differentcancer therapy protocol. Such a protocol can be selected from the groupconsisting of surgery, radiosurgery, chemotherapy, a treatmentcomprising administration of cytostatic(s), cytotoxic(s), a targetedtherapy, a vaccine, and any other biological or inorganic productintended to treat cancer.

Surprisingly, the herein described nanoparticles can further be used inthe context of radiotherapy alone with increased observed efficacy.

The invention can be used to treat any type of malignant tumor such ashaematological tumors or malignancies, and solid tumors, in particularof epithelial, neuroectodermal or mesenchymal origin. In addition,nanoparticles can be used to treat a premalignant lesion or a specificbenign disease where radiation therapy is classically used and/orindicated.

The invention is applicable, in the context of therapy, to primarytumors, or secondary invasions, loco-regional or distant metastases, andin the context of prophylaxis, in order to avoid secondary malignantcentral nervous system involvement such as the observed invasions(metastasis) from melanoma, lung cancer, kidney cancer, breast cancer,etc.

The nanoparticles can be used at any time throughout the anticancertreatment period. They can be administered for example as a neoadjuvant(before surgical intervention for cancer exeresis) or as an adjuvant(after surgery).

The nanoparticles can also be used for advanced tumors which cannot besurgically removed.

As herein explained, the irradiation can be applied at any time afteradministration of the particles, on one or more occasions, by using anycurrently available system of radiotherapy.

The nanoparticles herein described are in particular intended to be usedto treat cancer where radiotherapy is a classical treatment. Such cancermay be selected in particular from the group consisting of skin cancer,including malignant neoplasms associated to AIDS, melanoma; centralnervous system tumors including brain, stem brain, cerebellum,pituitary, spinal canal, eye and orbit; head and neck tumors; lungcancers; breast cancers; gastrointestinal tumors such as liver andhepatobiliary tract cancers, colon, rectum and anal cancers, stomach,pancreas, oesophagus cancer; male genitourinary tumors such as prostate,testis, penis and urethra cancers; gynecologic tumors such as uterinecervix, endometrium, ovary, fallopian tube, vagina and vulvar cancers;adrenal and retroperitoneal tumors; sarcomas of bone and soft tissueregardless the localization; lymphoma; myeloma; leukemia; and pediatrictumors such as Wilm's tumor, neuroblastoma, central nervous systemtumors, Ewing's sarcoma, etc.

The particles can be activated within a large range of total dose ofirradiation.

Amounts and schedules (planning and delivery of irradiations in a singledose, or in the context of a fractioned or hyperfractioned protocol,etc.) is defined for any disease/anatomical site/disease stage patientsetting/patient age (children, adult, elderly patient), and constitutesthe standard of care for any specific situation.

The irradiation can be applied at any time after administration of thenanoparticles, on one or more occasions, by using any currentlyavailable system of radiotherapy. The nanoparticles can be administeredby different routes such as local (intra-tumoral (IT) in particular),subcutaneous, intra venous (IV), intra-dermic, intra-arterial, airways(inhalation), intra peritoneal, intra muscular and oral route (per os).The nanoparticles can further be administered in an intracavity such asthe virtual cavity of tumor bed after tumorectomy.

Repeated injections or administrations can be performed, whenappropriate.

The term “treatment” denotes any action performed to correct abnormalfunctions, to prevent diseases, to improve pathological signs, such asin particular a reduction in the size or growth of an abnormal tissue,in particular of a tumor, a control of said size or growth, asuppression or destruction of abnormal cells or tissues, a slowing ofdisease progression, a disease stabilization with delay of cancerprogression, a reduction in the formation of metastases, a regression ofa disease or a complete remission (in the context of cancer forexample), etc.

As indicated previously, appropriate radiations or sources of ionizationare preferably ionizing radiations and can advantageously be selectedfrom the group consisting of X-Rays, gamma-Rays, electron beams, ionbeams and radioactive isotopes or radioisotopes emissions. X-Rays is aparticularly preferred source of ionization.

Ionizing radiations are typically of about 2 KeV to about 25 000 KeV, inparticular of about 2 KeV to about 6000 KeV (LINAC source), or of about2 KeV to about 1500 KeV (such as a cobalt 60 source). Using a X-Rayssource, particularly preferred ionizing radiations are typically ofabout 50 KeV to about 12 000 KeV, for example of about 50 KeV to about6000 KeV.

In general and in a non-restrictive manner, the following X-Rays can beapplied in different cases to activate the nanoparticles:

-   -   X-Rays of 50 to 150 keV which are particularly efficient for a        superficial target tissue;    -   X-Rays (ortho voltage) of 200 to 500 keV which can penetrate a        tissue thickness of 6 cm;    -   X-Rays (mega voltage) of 1000 keV to 25,000 keV. For example the        ionization of nanoparticles for the treatment of prostate cancer        can be carried out via five focused X-Rays with an energy of        15,000 keV.

Radioactive isotopes can alternatively be used as a ionizing radiationsource (named as curietherapy or brachytherapy). In particular, IodineI¹²⁵ (t ½=60.1 days), Palladium Pd¹⁰³ (t ½=17 days), Cesium Cs¹³⁷ andIridium Ir¹⁹² can advantageously be used.

Immunoradionuclide (or immunoradiolabelled ligand) can also be used as aionizing radiation source in the context of radioimmunotherapy. Suitableradionuclides for radioimmunotherapy may be, for example, selected from¹³¹I, ¹⁸⁶Re, ¹⁷⁷Lu or ⁹⁰Y.

Charged particles such as proton beams, ions beams such as carbon, inparticular high energy ion beams, can also be used as a ionizingradiation source and/or neutron beams.

Electron beams may also be used as a ionizing radiation source withenergy comprised between 4 MeV and 25 Mev.

Specific monochromatic irradiation source could be used in order toselectively generate X-rays with an energy close to or corresponding tothe desired X-ray absorption edge of the atom(s) of the metallicnanoparticle.

Preferentially sources of ionizing radiations may be selected fromLinear Accelerator (LINAC), Cobalt 60 and brachytherapy sources.

The term “in combination” indicates that the sought-after effect isobtained when the cells, tissues or organs of interest, being in contactwith the nanoparticles of the invention, are activated by the definedsource. However, it is not necessary for the particles and Rays to beadministered simultaneously, nor according to the same protocol.

The present disclosure further provides kits comprising any one or moreof the herein-described nanoparticles or compositions. Typically, thekit comprises at least one nanoparticle or population of nanoparticlesaccording to the present invention. Generally, the kit also comprisesone or more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s), a labelling notice providing instructions for using theproducts can be provided for using the nanoparticles, population ofnanoparticles or compositions according to the present methods.

Other aspects and advantages of the invention will become apparent inthe following examples, which are given for purposes of illustration andnot by way of limitation.

EXPERIMENTAL SECTION Example 1: Synthesis and Physico-ChemicalCharacterisation of Gold Nanoparticles with Different Sizes

Gold nanoparticles are obtained by reduction of gold chloride withsodium citrate in aqueous solution. Protocol was adapted from G. FrensNature Physical Science 241 (1973) 21.

In a typical experiment, HAuCl₄ solution is heated to boiling.Subsequently, sodium citrate solution is added. The resulting solutionis maintained under boiling for an additional period of 5 minutes.

The nanoparticle size is adjusted from 15 up to 105 nm by carefullymodifying the citrate versus gold precursor ratio (cf. Table 1).

The as prepared gold nanoparticles suspensions are then concentratedusing an ultrafiltration device (Amicon stirred cell model 8400 fromMillipore) with a 30 kDa cellulose membrane.

The resulting suspensions are ultimately filtered through a 0.22 μmcutoff membrane filter (PES membrane from Millipore) under laminar hoodand stored at 4° C.

Gold content is determined by ICP-MS and expressed as [Au] in g/L.

Particle size is determined using Transmission Electronic Microscopy(TEM) by counting more than 200 particles (FIG. 1A), taking the longestnanoparticle dimension for size measurement. Histograms are establishedand mean and standard deviation are reported (FIG. 1B).

TABLE 1 Particle Synthesis [Au] Samples size (nm) Citrate HauCl₄Structure g/L Gold-15 15 ± 2 (1σ) 20 mL 30 mM 500 mL 0.25 mM CFC 17.86Gold-30 32 ± 10 (1σ) 7.5 mL 40 mM 500 mL 0.25 mM CFC 16.90 Gold-60 60 ±10 (1σ) 2 mL 85 mM 500 mL 0.25 mM CFC 4.98 Gold-80 80 ± 10 (1σ) 1.2 mL43 mM 200 mL 0.30 mM CFC 10.67 Gold-105 105 ± 25 (1σ) 1.2 mL 39 mM 200mL 0.33 mM CFC 5.06Conclusion

The gold nanoparticles described in Table 1 are all prepared accordingto the same synthesis process to ensure the same surface characteristicproperties.

The TEM images show that the synthesized gold nanoparticles are allspherical and/or ovoid in shape.

The TEM electronic diffraction patterns show that all the synthesizedgold nanoparticles present a CFC structure.

Hence, only the size of the gold nanoparticle varies, with a wellcharacterized mean particle size and a size polydispersity in accordancewith prior art.

Example 2: Effect of Gold Nanoparticle Size on In Vitro Efficacy(Clonogenic Survival Assay) when the Gold Concentration is Constant atCellular Level

Protocol

To investigate the enhancement of the radiation response of the goldnanoparticles (GNPs) which are internalized into the cell or bound tothe cell (below expressed as gold concentration at cellular level),inventors used a specific clonogenic survival assay described below:

HT29 cells were plated at the density of 20 000 cells/cm². GNPs wereadded to the medium at various gold concentrations in the μM range.After an incubation time between 1 hours and 24 hours, the cellsurpernatants were removed. Then, the cells were washed briefly with PBSto remove all GNPs non-attached or non-internalized into the cells.Inventors next performed cell trypsination and counted the cell numberusing Haemocytometer.

For each condition, inventors took a sample of 100000 cells/mL up to220000 cells/mL further analysed for gold concentration by ICP-MS.

Gold (Au) concentration at cellular level (number of gold atoms pervolume) is expressed in μM.

This parameter may also be expressed in term of gold (Au) concentrationper target cell as follow:cell:Au=1:X (X expressed in nmole)according to the following calculation:

  Cell = 1${X\left( {{expressed}\mspace{14mu}{in}\mspace{14mu}{nmole}} \right)} = \frac{{Gold}\mspace{14mu}({Au})\mspace{14mu}{concentration}\mspace{14mu}({µM}) \times 1\mspace{14mu}({mL})}{1000\mspace{14mu}({mL}) \times {Number}\mspace{14mu}{of}\mspace{14mu}{cells}\mspace{14mu}{per}\mspace{14mu}{mL}}$

The other cells were plated (at the density of 300 up to 1000cells/wells according to the treatments conditions) to performed theclonogenic assays. Once the cells were attached to the plate, they wereeither non irradiated (sham control), or irradiated with doses of 2 Gyand 4 Gy using a 200 kVp X-Ray device. The cells were allowed to grow toform colonies up to 12 days. Then, the colonies were fixed and stainedwith crystal violet and counted in order to estimate clonogenic survivalfraction (SF) (See FIGS. 3A and 3B) by using the following formulae:

Plating efficiency (PE) is the ratio of the number of colonies formedwithout any irradiation to the number of seeded cells:PE=no of colony formed×100/no seeded cells

Surviving fraction (SF) represents the level of viable cells afterirradiation and is normalized to the PE of the control:SF=no colonies formed after treatment/(no cells seeded*PE)

Dose Enhancement Factor (DEF) is estimated as the ratio of SF (radiationdose alone)/SF (gold nanoparticles activated with the same radiationdose).

TABLE 2 Gold (Au) concentration at cellular level (μM) Gold (Au)concentration per target cell Cell: Au = 1:X (X expressed in nmole)Number of GOLD [Au] Cells Cell: Au = SAMPLES μM per mL 1:X (nmole)GOLD-15 GOLD-30 GOLD-60 GOLD-80 GOLD-105 6 1.6 × 10⁵  3.6 × 10⁻⁵ GOLD-1512 1.3 × 10⁵  9.5 × 10⁻⁵ GOLD-30 17 1.3 × 10⁵  13.1 × 10⁻⁵ GOLD-60 162.2 × 10⁵  7.3 × 10⁻⁵ GOLD-80 17 2.0 × 10⁵  8.5 × 10⁻⁵ GOLD-105 17 1.3 ×10⁵  13.6 × 10⁻⁵ GOLD-15 20 9.7 × 10⁴  20.7 × 10⁻⁵ GOLD-30 40 1.8 × 10⁵ 22.6 × 10⁻⁵ GOLD-60 83 2.2 × 10⁵  37.7 × 10⁻⁵ GOLD-80 52 2.1 × 10⁵ 24.9 × 10⁻⁵ GOLD-105 59 1.4 × 10⁵  43.4 × 10⁻⁵ GOLD-15 130 1.4 × 10⁵ 92.0 × 10⁻⁵ GOLD-30 148 1.4 × 10⁵ 108.3 × 10⁻⁵ GOLD-60 95 1.5 × 10⁵ 61.9 × 10⁻⁵ GOLD-80 119 1.8 × 10⁵  65.4 × 10⁻⁵ GOLD-15 GOLD-30 GOLD-60400 2.2 × 10⁵ 181.8 × 10⁻⁵ GOLD-80 GOLD-105Table 2 shows gold concentration at cellular level (μM) [or goldconcentration per target cell (cell:Au=1:X (X expressed in nmole))] fordifferent gold (Au) concentrations incubated with HT29 cancer cells foreach gold nanoparticles synthesized in Example 1, Table 1.

TABLE 3A Gold (Au) concentration at cellular level (μM) Gold (Au)concentration per target cell Cell: Au = 1:X (X expressed in nmole)Number GOLD [Au] of cells Cell: Au = DEF SAMPLES μM per mL 1:X (nmole)(4 Gy) GOLD-15 12 1.3 × 10⁵  9.5 × 10⁻⁵ 0.98 GOLD-30 17 1.3 × 10⁵ 13.1 ×10⁻⁵ 0.99 GOLD-60 16 2.2 × 10⁵  7.3 × 10⁻⁵ 1.09 GOLD-80 17 2.0 × 10⁵ 8.5 × 10⁻⁵ 0.98 GOLD-105 17 1.3 × 10⁵ 13.6 × 10⁻⁵ 1.35Table 3A reports the DEF values obtained for a 4 Gy irradiation dose, ofthe gold nanoparticles described in example 1, when gold concentrationat cellular level is below 20 μM [or corresponding gold concentrationper target cell is below 15*10⁻⁵ nmole (Cell:Au≤1:15*10⁻⁵ nmole)].

TABLE 3B Gold (Au) concentration at cellular level (μM) Gold (Au)concentration per target cell Cell: Au = 1:X (X expressed in nmole)Number GOLD [Au] of cells Cell: Au = DEF SAMPLES μM per mL 1:X (nmole)(4 Gy) GOLD-15 20 9.7 × 10⁴ 20.7 × 10⁻⁵ 0.96 GOLD-30 40 1.8 × 10⁵ 22.6 ×10⁻⁵ 1.16 GOLD-60 83 2.2 × 10⁵ 37.7 × 10⁻⁵ 1.13 GOLD-80 52 2.1 × 10⁵24.9 × 10⁻⁵ 1.63 GOLD-105 59 1.4 × 10⁵ 43.4 × 10⁻⁵ 2.59Table 3B reports the DEF values obtained for a 4 Gy irradiation dose, ofthe gold nanoparticles described in example 1, when gold concentrationat cellular level is between 20 μM and 83 μM [or corresponding goldconcentration per target cell is between 20×10⁻⁵ nmole and 45×10⁻⁵ nmole(1:20×10⁻⁵ nmole≤Cell:Au≤1:45×10⁻⁵ nmole)].

TABLE 3C Gold (Au) concentration at cellular level (μM) Gold (Au)concentration per target cell Cell: Au = 1:X (X expressed in nmole)Number GOLD [Au] of cells Cell: Au = DEF SAMPLES μM per mL 1:X (nmole)(4 Gy) GOLD-15 130 1.4 × 10⁵  92.0 × 10⁻⁵ 1.57 GOLD-30 148 1.4 × 10⁵108.3 × 10⁻⁵ 1.23 GOLD-60 95 1.5 × 10⁵  61.9 × 10⁻⁵ 1.63 GOLD-80 119 1.8× 10⁵  65.4 × 10⁻⁵ 3.36Table 3C reports the DEF values obtained for a 4 Gy irradiation dose, ofthe gold nanoparticles described in example 1, when gold concentrationat cellular level is between 95 μM and 148 μM [or corresponding goldconcentration per target cell is between 60×10⁻⁵ nmole and 110×10⁻⁵nmole (1:60×10⁻⁵ nmole≤Cell:Au≤1:110×10⁻⁵ nmole)].Conclusion

Surprisingly, a threshold in the DEF value is observed for goldnanoparticles with particle size ≥80 nm (see FIGS. 5A and 5B).

Example 3: Effect of Gold Nanoparticle Size on In Vitro Efficacy whenthe X-Ray Attenuation Capacity of Each Tested Gold Nanoparticle isConstant at Cellular Level

Protocol: X-Ray Attenuation Measurement

Gold nanoparticles with different gold concentration (expressed in [Au]g/L) were prepared in 200 μL tubes and placed in a custom-designedpolystyrene holder.

ρCT was performed using a General Electric Locus ρCT system with anodevoltage and current of respectively 50 KV and 450 ρA.

Scanning was performed using a 90 μm isotropic resolution mode.

A cylindrical small region of interest was carefully placed in the 3Dimage over the center of each tube to measure attenuation values offluid-filled tubes containing gold nanoparticles dispersions.

Conclusion

A similar X-rays attenuation value is observed whatever the goldnanoparticle size, for size comprised between 15 nm and 105 nm (seeFIGS. 6A and 6B). This result confirms the threshold effect on efficacyobserved for a nanoparticle size 80 nm. Such a nanoparticle is able togenerate more damages at cellular level for a given absorbed X-Rayenergy (see FIGS. 4A and 4B).

Example 4: Effect of Gold Nanoparticle Localization at Cellular Level(Physical Interaction with Tumor Cell Membranes and/or Cell Uptake) onIn Vitro Efficacy

HT29 cells are plated with the appropriate cells number to form between50 and 200 colonies according to the treatment. When cells are attached,50 μM, 100 μM or 400 μM of gold are added with an incubation time ofless than 5 minutes (no incubation) or 12 hours. Gold nanoparticle withparticle size of 60 nm (GOLD-60 from example 1) were tested. Cells wereeither non irradiated (sham control), or irradiated with doses of 2 Gyand 4 Gy using a 200 kVp X-Ray device. After irradiation, cells wereincubated between 10 to 12 days at 37° C. The clones were fixed andstained with crystal violet and counted to estimate clonogenic survivalfraction.

FIG. 7A shows the surviving fraction at 4 Gy (SF4) of HT29 cellsincubated with gold nanoparticles (GOLD-60 from example 1) for less than5 minutes, and FIG. 7B shows the surviving fraction at 4 Gy (SF4) ofHT29 cells incubated with gold nanoparticles (GOLD-60 from example 1)for about 12 hours.

TABLE 4 DEF GOLD-60 50 μM 100 μM 400 μM 4 Gy, incubation time less than5 mn 0.96 1.25 1.8 4 Gy, incubation time 12 hours 0.88 1 1.5Table 4 presents the DEF of gold nanoparticle (GOLD-60) for goldconcentration of 50 μM, 100 μM and 400 μM, at 4 Gy, for an incubationtime of less than 5 mn or of 12 hours.Conclusion

The data show, for a gold concentration of 400 μM, similar significantDEF values for gold nanoparticles incubated less than 5 minutes and forgold nanoparticles incubated 12 hours with cells prior irradiation.

These results demonstrate that gold nanoparticles (GNPs) enhance thetarget cell radiation response without it being necessary for the GNPsto be internalized by the cell. Indeed, and as known by the man of theart, two hours are necessary to allow the cell uptake of about 50% ofthe nanoparticles present in the biological medium (see Chitrani et al.,2006, for example).

When gold nanoparticles are in contact with cancer cells they areadvantageously able to induce cancer cell damages under irradiation.

The above experimental results of examples 1 to 4 highlight the abilityof metallic nanoparticles to induce an enhanced therapeutic effect whenadministered in vivo if an adequate metal concentration (proportional tothe tumor weight as apparent to the man of the art) is present on thetumor site (the metallic nanoparticles cell uptake is not required asdemonstrated previously).

A significantly smaller amount of metal is required per target cell,when using metallic nanoparticles with a particle size ≥80 nm whencompared to the metal amount required when using metallic nanoparticleswith a particle size of about 60 nm, to produce an efficient therapeuticeffect when metallic nanoparticles are exposed to ionizing radiations(via radiotherapy for example).

In order to produce an efficient therapeutic effect under ionizingradiations, the use of metallic nanoparticles with a nanoparticle size≥80 nm requires a metal concentration per target cell which is betweenabout 2 and 7 times, in particular between 4 and 7 times or between 2and 5 times, inferior to the metal concentration per target cellrequired when using metallic nanoparticles with a nanoparticle size ofabout 60 nm or less.

Such metallic nanoparticles appear advantageous for in vivo uses.

A population of metallic nanoparticles wherein the mean largest size ofa nanoparticle of the population is between 80 and 105 nm, isparticularly advantageous in therapy when said nanoparticles are exposedto ionizing radiations. Such nanoparticles indeed, in particular, allowan enhanced Dose Enhancement Factor (DEF). A threshold effect isobserved in vitro for a nanoparticle as herein described the largestsize of which is preferably 80 nm, and even more preferably betweenabout 80 nm and 105 nm. Such a nanoparticle exhibits a reduced surfacearea allowing an improved biocompatibility and in consequence a reducedtoxicity.

A population of metallic nanoparticles as herein described furtherallows a reduced tumor clearance. A single injection of a compositionaccording to the present invention now allows the required therapeuticeffect in the context of a multi fractionated irradiation protocol ascurrently applied in clinic.

REFERENCES

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We claim:
 1. A method for inducing in vitro, ex vivo or in vivo theperturbation, lysis or destruction of target human cells selected fromthe group consisting of benign cells, pre-malignant cells and malignantcells, comprising i) contacting said target cells with a population ofmetallic nanoparticles during a period of time sufficient to allow thenanoparticles to interact with said cells, each nanoparticle of thepopulation being made of a metal having an atomic number (Z) of at least25, each metallic nanoparticle of the population being covered with abiocompatible coating, and the mean largest size of the nanoparticles ofthe population being between 80 and 105 nm, and, ii) exposing the cellsto ionizing electromagnetic radiation, said exposure inducing or causingthe perturbation, lysis or destruction of said cells.
 2. The methodaccording to claim 1, wherein the metal is selected from gold (Au),Silver (Ag), platinum (Pt), palladium (Pd), tin (Sn), Zirconium (Zr), orIron (Fe).
 3. The method according to claim 2, wherein the population ofmetallic nanoparticles comprises between 10⁻⁶ nmole and 10⁻³ nmole ofmetal per target cell.
 4. The method according to claim 1, wherein saidmetallic nanoparticles comprises a surface component enabling specifictargeting of biological tissues or cells.
 5. The method according toclaim 1, wherein each metallic nanoparticle is essentially spherical orovoid in shape.
 6. The method according to claim 1, wherein saidionizing electromagnetic radiation is selected from the group consistingof X-rays and γ-rays.
 7. The method according to claim 6, wherein saidionizing electromagnetic radiation is between 50 KeV to 12 000 KeV. 8.The method according to claim 6, wherein said ionizing electromagneticradiation is X-ray radiation between 50 KeV to 6000 KeV.
 9. The methodaccording to claim 1, wherein said malignant cells are cells from asolid tumor.
 10. The method according to claim 1, wherein said targethuman cells are also exposed to an additional therapeutic compound,distinct from the population of metallic nanoparticles, intended totreat cancer.
 11. The method according to claim 1, wherein thebiocompatible coating is a non-biodegradable coating selected from thegroup consisting of silica, alumina, sugar, phosphate, silane, thiol,zwitterionic compound, lipid, saturated carbon polymer and an inorganicpolymer; or a biodegradable coating selected from the group consistingof biological polymer, phospholipid, saccharide, oligosaccharide andpolysaccharide.
 12. A method for treating a disorder or alleviatingsymptoms of the disorder, in a human patient having abnormal cells,comprising i) administering to the human patient suffering from thedisorder a population of nanoparticles, in conditions allowing thenanoparticles to interact with the abnormal cells, each nanoparticle ofthe population being made of a metal having an atomic number (Z) of atleast 25, each metallic nanoparticle of the population being coveredwith a biocompatible coating, and the mean largest size of thenanoparticles of the population being between 80 and 105 nm and ii)subsequently treating the patient by exposing said patient to ionizingelectromagnetic radiation, said exposition leading to an alteration,disturbance or functional destruction of the patient's abnormal cells,thereby preventing or treating the disorder or alleviating the symptomsof the disorder.
 13. A pharmaceutical composition intended to alter ordestroy target cells in a human when said cells are exposed to ionizingelectromagnetic radiation, said pharmaceutical composition comprising apopulation of metallic nanoparticles and a pharmaceutically acceptableexcipient, wherein each nanoparticle is made of a metal having an atomicnumber (Z) of at least 25, and the mean largest size of thenanoparticles of the population is between 80 and 105 nm.